Turchi J J, Henkels K M, Zhou Y
Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA.
Nucleic Acids Res. 2000 Dec 1;28(23):4634-41. doi: 10.1093/nar/28.23.4634.
We have determined the effect of cisplatin-DNA damage on the ability of the DNA-dependent protein kinase (DNA-PK) to interact with duplex DNA molecules in vitro. The Ku DNA binding subunits of DNA-PK display a reduced ability to translocate on duplex DNA containing cisplatin-DNA adducts compared to control, undamaged duplex DNA. The decreased rates of translocation resulted in a decrease in the association of the p460 catalytic subunit of DNA-PK (DNA-PKcs) with the Ku-DNA complex. In addition to a decrease in DNA-PKcs association, the DNA-PKcs that is bound with Ku at a DNA end containing cisplatin-DNA adducts has a reduced catalytic rate compared to heterotrimeric DNA-PK assembled on undamaged DNA. The position of the cisplatin-DNA lesion from the terminus also effects kinase activation, with maximal inhibition occurring when the lesion is closer to the terminus. These results are consistent with a model for DNA-PK activation where the Ku dimer translocates away from the DNA terminus and facilitates the association of DNA-PKcs which interacts with both Ku and DNA resulting in kinase activation. The presence of cisplatin adducts decreases the ability to translocate away from the terminus and results in the formation of inactive kinase complexes at the DNA terminus. The results are discussed with respect to the ability of cisplatin to sensitize cells to DNA damage induced by ionizing radiation and the ability to repair DNA double-strand breaks.
我们已经确定了顺铂-DNA损伤对DNA依赖性蛋白激酶（DNA-PK）在体外与双链DNA分子相互作用能力的影响。与对照的未损伤双链DNA相比，DNA-PK的Ku DNA结合亚基在含有顺铂-DNA加合物的双链DNA上的易位能力降低。易位速率的降低导致DNA-PK的p460催化亚基（DNA-PKcs）与Ku-DNA复合物的结合减少。除了DNA-PKcs结合减少外，与组装在未损伤DNA上的异源三聚体DNA-PK相比，在含有顺铂-DNA加合物的DNA末端与Ku结合的DNA-PKcs的催化速率降低。顺铂-DNA损伤距末端的位置也影响激酶激活，当损伤更靠近末端时，抑制作用最大。这些结果与DNA-PK激活模型一致，即Ku二聚体从DNA末端移位并促进与Ku和DNA都相互作用的DNA-PKcs的结合，从而导致激酶激活。顺铂加合物的存在降低了从末端移位的能力，并导致在DNA末端形成无活性的激酶复合物。讨论了这些结果与顺铂使细胞对电离辐射诱导的DNA损伤敏感的能力以及修复DNA双链断裂的能力的关系。
